The present invention is directed to fluorescent dyes with increased brightness and methods of use thereof.
Fluorescent dyes conjugated to antibodies are commonly used for immunofluorescence analysis. A vast number of variants in antibodies, fluorescent dyes, flow cytometers, flow sorters and fluorescence microscopes has been developed in the last two decades to enable specific detection and isolation of target cells. One issue in immunofluorescence technology is the detection threshold of the fluorescence emission, which can be enhanced, for example, by better detectors, filter systems or modified fluorescent dyes.
A limitation of conventional small molecule fluorescent dye molecules is their limited brightness. Therefore biomolecules are typically labeled with multiple dye molecules to increase the brightness of the fluorochrome conjugate. Most of the aforementioned fluorescent dye molecules, such as rhodamines or cyanines, contain planar aromatic chromophores, which are prone to hydrophobic interactions leading to dye-dye dimers with low or no fluorescence. Consequently, fluorescence intensity of labeled biomolecules is not proportional to the degree of fluorescent dye labeling of said biomolecules. At higher degrees of labeling (DOLs) the fluorescence intensity of the single dye might even decrease due to self-quenching mechanism caused by dimer, trimer or multimer formation.
These undesired formations have been reduced to some extent by adding substituents to the flat aromatic dye molecules, which increase water solubility. Suitable substituents described in the literature might impart charges to the dye molecule, such as sulfonate groups described, e.g., in U.S. Pat. Nos. 5,268,486 and 6,977,305, 6,130,101 and Panchuk-Voloshina, et al., J. Histochem. Cytochem. 47(9), 1179 (1999), or phosphate groups described, e.g., in WO2013056720. Other suitable dimerization reducing substituents are bulky water-soluble polymers, such as polyethylene glycol described, e.g., in patent application WO2009078970, or charged dendrimers described, e.g., in U.S. Pat. Nos. 6,913,743, and 7,655,217. Although biomolecule conjugates of these substituted dyes achieve higher brightness at higher degrees of labeling (DOLs) than conjugates of the unsubstituted parent dyes, there is still a deviation from a linear proportionality between fluorescence intensity and degree of labeling of the labeled biomolecule albeit at a higher DOL as for the unsubstituted parent dye.
Conjugate brightness is also limited by the number of functionalization sites available on the biomolecule, which can be functionalized without loss of biomolecule activity. As a result DOLs of biomolecule conjugates are still limited, e.g., for antibodies (IgG) they are typically in the range of 4 to 8 in the case of hydrophilic labels (R. P. Haugland, Current Protocols in Cell Biology (2000) 16.5.1-16.5.22). Consequently the brightness of these conjugates is still inferior to, e.g., conjugates of phycobiliproteins, such as phycoerythrin (PE) or allophycocyanine (APC).
The high fluorescence intensity of phycobiliproteins and their biomolecule conjugates is due to the presence of multiple fluorophore subunits within a phycobiliprotein. R-phycoerythrin, e.g., contains 34 phycobilin fluorophore subunits (A. N. Glazer, J. Appl. Phycol. 6, 105 (1994)). Therefore biomolecule conjugates of phycobiliproteins, such as PE or APC are popular, e.g., in flow cytometry, despite their drawbacks stemming from their protein nature such as limited stability against non-physiological solvents, temperature, and pH values as well as their limited photostability and their generally limited shelf life. Another drawback is the limited availability of different colors, which limit multiplexing capabilities within a single fluorescence assay.
There have been efforts to construct multichromophore constructs with phycobiliprotein-like fluorescence properties by arranging fluorophores into precise supramolecular structures preventing self-quenching of excited fluorophores. Benvin et al., J. Am. Chem. Soc. 129(7), 2025 (2007) describe fluorescent dyes intercalated into supramolecular DNA templates. However, a drawback of this method is the non-covalent binding of the fluorophores within the DNA scaffold. Migration of dye molecules out of the DNA scaffold can lead to loss of fluorescence signal. In case of a multicolor experiment dye exchange between differently labeled DNA scaffolds might lead to false positive fluorescent signals. In case of a multiparameter experiment employing multiple colors it is advisable to use covalently bound fluorophores.
Another class of brightly fluorescent dyes for biomolecule labeling are fluorescent polymers based on semiconducting polymers, such as polyfluorenes described e.g. in U.S. Pat. Nos. 8,158,444, 8,354,239, and 8,802,450 and 8,362,193, 8,455,613, and 8,575,303. These polymers also contain multiple fluorophore subunits, as the effective conjugation length within the polymer is limited to 9-10 monomer subunits.
Preparing brighter fluorescent dyes by multimerizing conventional fluorescent dye molecules on a water-soluble scaffold has so far not resulted in compounds useful for the labeling of biomolecules. Recommendations for fluorescent labeling of, e.g., dextrans are 0.3-0.7 dye molecules per dextran in the 3000 MW range, 0.5-2 dye molecules in the 10,000 MW range, 2-4 dye molecules in the 40,000 MW range and 3-6 dye molecules in the 70,000 MW range. Due to their large size and bulkiness these dye multimers offer no benefit in biomolecule labeling and are unsuitable for preparing biomolecule conjugates with phycobiliprotein-like fluorescence intensities. Fluorescently labeled dextrans with higher degree of labeling show quenching due to dye-dye interaction. This has found use in U.S. Pat. No. 5,719,031, wherein the degree of labeling of dextran-fluorochrome-conjugates is high enough to furnish fluorescent quenching. Enzymatic degradation of the dextran-fluorochrome-conjugate is accompanied by an enhancement of the fluorescence emission signal, which is used for quantification of the enzymatic digestion process.
There remains still a considerable need for improved fluorescent dyes for labeling of biomolecules, which provide phycobiliprotein-like fluorescence intensity without the limitations of phycobiliproteins and fluorescent polymers.